This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Steroidogenic acute regulatory protein (StAR) is a 37 kDa protein that simulates steroidogenesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane where it is converted to pregnenolone by P450scc. StAR acts exclusively on the OMM and partially unfolds to a molten globule at pH 3.5 ~ 4.0;it has been suggested, but not proven, that this molten globule transition is required for activity. The C-terminal helix interacts with the OMM, as liposomes composed of OMM lipids protect only this helix from proteolysis. Modeling shows that this C-helix, which forms the floor of the sterol-binding pocket (SBP), is stabilized by hydrogen bonding to adjacent loops. We subjected our model of StAR to molecular dynamics for 3 nsec at 300K using AMBER 7.0 under two conditions: default settings and protonation of D, E and H (to mimic pH 4.0). Trajectory analysis shows the W1 loop and C-helix are much more mobile at pH 4, opening and closing the SBP. We tested the effect of this movement on StAR activity by designing two disulfide mutants linking the C- helix to the W1 loop. Modeling showed that neither of these paired mutants, D106C/A268C (DA) and S100C/S261C (SS), disrupted StAR folding or the size and shape of the SBP. The DA and SS mutants were expressed with an intein vector and purified. MS analysis confirmed the locations of the disulfides. DA lost all cholesterol-binding capacity and steroidogenic activity with isolated mitochondria in vitro, and SS lost ~50% of both. Full binding and activity was restored to each by disrupting the disulfides with DTT. These data support the model that StAR activity requires a pH-dependant transition to a molten globule on the OMM.